WO2016054231A1 - Highly potent acid alpha-glucosidase with enhanced carbohydrates - Google Patents

Highly potent acid alpha-glucosidase with enhanced carbohydrates Download PDF

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Publication number
WO2016054231A1
WO2016054231A1 PCT/US2015/053252 US2015053252W WO2016054231A1 WO 2016054231 A1 WO2016054231 A1 WO 2016054231A1 US 2015053252 W US2015053252 W US 2015053252W WO 2016054231 A1 WO2016054231 A1 WO 2016054231A1
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rhgaa
composition
glycans
gaa
atb
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PCT/US2015/053252
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English (en)
French (fr)
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Russell GOTSCHALL
Hung Do
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Amicus Therapeutics, Inc.
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Priority to IL307587A priority Critical patent/IL307587A/en
Priority to CA2961762A priority patent/CA2961762C/en
Priority to SG11201702114TA priority patent/SG11201702114TA/en
Priority to MA40150A priority patent/MA40150A1/fr
Priority to FIEP15845664.0T priority patent/FI3201320T3/fi
Priority to TN2017000082A priority patent/TN2017000082A1/en
Priority to EA201790724A priority patent/EA038986B1/ru
Priority to DK15845664.0T priority patent/DK3201320T3/da
Priority to MX2017003989A priority patent/MX2017003989A/es
Priority to HRP20240041TT priority patent/HRP20240041T1/hr
Priority to KR1020217030450A priority patent/KR102455814B1/ko
Priority to BR112017005810A priority patent/BR112017005810A2/pt
Application filed by Amicus Therapeutics, Inc. filed Critical Amicus Therapeutics, Inc.
Priority to KR1020177011346A priority patent/KR102306577B1/ko
Priority to MYPI2017000365A priority patent/MY186336A/en
Priority to CN201580052512.5A priority patent/CN107075468B/zh
Priority to KR1020227035610A priority patent/KR20220146662A/ko
Priority to EP23191307.0A priority patent/EP4273241A3/en
Priority to JP2017516917A priority patent/JP6851964B2/ja
Priority to NZ729507A priority patent/NZ729507B2/en
Priority to RS20240027A priority patent/RS65066B1/sr
Priority to PL15845664.0T priority patent/PL3201320T3/pl
Priority to IL289383A priority patent/IL289383B2/en
Priority to CN202111483006.5A priority patent/CN114540327A/zh
Priority to CR20170107A priority patent/CR20170107A/es
Priority to SI201531983T priority patent/SI3201320T1/sl
Priority to US15/515,808 priority patent/US10208299B2/en
Priority to AU2015325028A priority patent/AU2015325028B2/en
Priority to LTEPPCT/US2015/053252T priority patent/LT3201320T/lt
Priority to EP15845664.0A priority patent/EP3201320B1/en
Publication of WO2016054231A1 publication Critical patent/WO2016054231A1/en
Priority to PH12017500455A priority patent/PH12017500455A1/en
Priority to IL251152A priority patent/IL251152B/en
Priority to ZA2017/01945A priority patent/ZA201701945B/en
Priority to CONC2017/0002776A priority patent/CO2017002776A2/es
Priority to US16/252,505 priority patent/US10961522B2/en
Priority to IL277529A priority patent/IL277529B/en
Priority to US17/249,175 priority patent/US11753632B2/en
Priority to US17/665,179 priority patent/US11591583B2/en
Priority to AU2022203498A priority patent/AU2022203498A1/en
Priority to US18/111,321 priority patent/US20230203465A1/en
Priority to FIC20240010C priority patent/FIC20240010I1/fi

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention involves the fields of medicine, genetics and recombinant glycoprotein biochemistry, and, specifically, relates to recombinant human alpha glucosidase (rhGAA) compositions that have a higher total content of mannose 6-phosphate-bearing glycans that efficiently target CIMPR on muscle cells and subsequently deliver rhGAA to the lysosomes where it can break down abnormally high levels of accumulated glycogen.
  • the rhGAA of the invention exhibits superior targeting to muscle cells and subsequent delivery to lysosomes compared to conventional rhGAA products and exhibits other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy of subjects having Pompe disease.
  • “Lumizyme” and “Myozyme” are conventional forms of rhGAA produced or marketed as biologies by Genzyme and approved by the U.S. Food and Drug Administration and are described by reference to the Physician 's Desk Reference (2014)(which is hereby
  • Alglucosidase Alfa is identified as chemical name [ 199-arginine,223 -histidine]prepro-a-glucosidase (human);
  • Pompe disease is an inherited lysosomal storage disease that results from a deficiency in acid a-glucosidase (GAA) activity.
  • a person having Pompe Disease lacks or has reduced levels of acid alpha-glucosidase (GAA), the enzyme which breaks down glycogen, and a substance the body uses as an energy source.
  • GAA acid alpha-glucosidase
  • This enzyme deficiency causes excess glycogen accumulation in the lysosomes, which are intra-cellular organelles containing enzymes that ordinarily break down glycogen and other cellular debris or waste products. Glycogen accumulation in certain tissues of a subject having Pompe Disease, especially muscles, impairs the ability of cells to function normally.
  • glycogen In Pompe Disease, glycogen is not properly metabolized and progressively accumulates in the lysosomes, especially in skeletal muscle cells and, in the infant onset form of the disease, in cardiac muscle cells. The accumulation of glycogen damages the muscle and nerve cells as well as those in other affected tissues
  • Pompe disease is clinically recognized as either an early infantile form or as a late onset form.
  • the age of onset tends to parallel the severity of the genetic mutation causing Pompe Disease.
  • the most severe genetic mutations cause complete loss of GAA activity manifest as early onset disease during infancy.
  • Genetic mutations that diminish GAA activity but do not completely eliminate it are associated with forms of Pompe disease having delayed onset and progression.
  • Infantile onset Pompe disease manifests shortly after birth and is characterized by muscular weakness, respiratory insufficiency and cardiac failure. Untreated, it is usually fatal within two years. Juvenile and adult onset Pompe disease manifest later in life and usually progress more slowly than infantile onset disease. This form of the disease, while it generally does not affect the heart, may also result in death, due to weakening of skeletal muscles and those involved in respiration.
  • ERT enzyme replacement therapy
  • rhGAA recombinant human GAA
  • Lumizyme® recombinant human GAA
  • the rhGAA is administered in an attempt to replace or supplement the missing or defective GAA in a subject having Pompe Disease.
  • most of the rhGAA in conventional rhGAA products does not target muscle tissue it is non-productively eliminated after administration. This occurs because conventional rhGAAs lack a high total content of M6P- and bis- M6P-bearing glycans which target a rhGAA molecule to the CIMPR on target muscle cells where it is subsequently transported into the cell's lysosomes.
  • M6P mannose-6- phosphate
  • CIMPR cation-independent mannose 6-phosphate receptor
  • rhGAA There are seven potential N-linked glycosylation sites on rhGAA. Since each glycosylation site is heterogeneous in the type of N-linked oligosaccharides (N-glycans) present, rhGAA consist of a complex mixture of proteins with N-glycans having varying binding affinities for M6P receptor and other carbohydrate receptors. rhGAA that contains a high mannose N-glycans having one M6P group (mono-M6P) binds to CIMPR with low (-6,000 nM) affinity while rhGAA that contains two M6P groups on same N-glycan (bis- M6P) bind with high ( ⁇ 2 nM) affinity.
  • mono-M6P mono-M6P
  • bis- M6P bis- M6P
  • Fig. 1A Representative structures for non-phosphorylated, mono-M6P, and bis-M6P glycans are shown by Fig. 1A.
  • the mannose-6-P group is shown by Fig. IB.
  • rhGAA can enzymatically degrade accumulated glycogen.
  • conventional rhGAAs have low total levels of M6P- and bis-M6P- bearing glycans and, thus, target muscle cells poorly resulting in inferior delivery of rhGAA to the lysosomes.
  • the majority of rhGAA molecules in these conventional products do not have phosphorylated N-glycans, thereby lacking affinity for the CIMPR.
  • Non-phosphorylated high mannose glycans can also be cleared by the mannose receptor which results in nonproductive clearance of the ERT (Fig. 2).
  • N-glycans complex carbohydrates, which contain, galactose and sialic acids are also present on rhGAA. Since complex N-glycans are not phosphorylated they have no affinity for CIMPR. However, complex -type N-glycans with exposed galactose residues have moderate to high affinity for the asialoglycoprotein receptor on liver hepatocytes which leads to rapid non-productive clearance of rhGAA (Fig 2).
  • glycosylation of GAA or rhGAA can be enzymatically modified in vitro by the phosphotransferase and uncovering enzymes described by Canfield, et al., U.S. Patent No. 6,534,300, to generate M6P groups. Enzymatic glycosylation cannot be adequately controlled and produces rhGAA having undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high-mannose N-glycans which all could be potentially enzymatically phosphorylated in vitro with a
  • phosphotransferase/uncovering enzyme may contain on average 5-6 M6P groups per GAA.
  • the glycosylation patterns produced by in vitro enzymatic treatment of GAA are problematic because the additional terminal mannose residues, particularly non- phosphorylated terminal mannose residues, negatively affect the pharmacokinetics of the modified rhGAA.
  • these mannose groups increase non-productive clearance of the GAA, increase the uptake of the enzymatically-modified GAA by immune cells, and reduce rhGAA therapeutic efficacy due to less of the GAA reaching targeted tissues, such as cardiac or skeletal muscle myocytes.
  • terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen which leads to rapid clearance of the
  • glycosylation pattern of enzymatically-modified GAA having high mannose N-glycans with terminal non-phosphorylated mannose residues resembles that on glycoproteins produced in yeasts, molds and function increasing the risk of triggering immune or allergic responses, such as life-threatening severe allergic (anaphylactic) or hypersensitivity reactions, to the enzymatically modified rhGAA.
  • a large portion of the GAA in a conventional rhGAA does not contain glycans bearing mono- or bis-M6P, which target the rhGAA to muscle cells.
  • a subject's immune system is exposed to this excess non-phosphorylated GAA and can generate detrimental immune responses that recognize GAA.
  • Induction of an immune responses to the non-phosphorylated GAA that does not enter the target tissues and deliver to the lysosomes increase the risk of treatment failure due to immunological inactivation of the administered rhGAA and increases the risk of the patient experiencing detrimental autoimmune or allergic reactions to the rhGAA treatment.
  • the rhGAA according to the invention contains significantly less of this non-targeted, non-phosphorylated rhGAA, thus reducing exposure of a patient's immune system to it.
  • rhGAA' s contain a higher content of non-phosphorylated rhGAA which does not target the CIMPR on muscle cells.
  • rhGAA that does not bind to CIMPR on muscle cells and then enter the lysosome does not enzymatically degrade glycogen there.
  • equivalent doses of a conventional rhGAA and the rhGAA according to the invention are administered, more rhGAA in the composition according to the invention binds CIMPR on muscle cells and then delivers to the lysosome.
  • the rhGAA of the invention provides a doctor with the option of administering a lower amount of rhGAA while delivering the same or more rhGAA to the lysosome.
  • Myozyme®, Lumizyme® or Alglucosidase Alfa have not significantly increased the content of M6P or bis-M6P because cellular carbohydrate processing is naturally complex and extremely difficult to manipulate.
  • the inventors diligently sought and identified ways to efficiently target rhGAA to muscle cells and deliver it to the lysosome, minimize non-productive clearance of rhGAA once administered, and thus more productively target rhGAA to muscle tissue.
  • rhGAA of the invention has well-processed complex-type N-glycans which minimize non-productive clearance of the rhGAA by non-target tissues.
  • rhGAA in CHO cells having significantly higher total content of mono-M6P and bis-M6P glycans which target CIMPR on muscle cells and then deliver the rhGAA to the lysosomes.
  • the rhGAA produced by this method also has advantageous pharmacokinetic properties by virtue of its overall glycosylation pattern that increases target tissue uptake and decreases non-productive clearance following administration to a subject having Pompe Disease.
  • the inventors show that the rhGAA of the invention, as exemplified by rhGAA designated as ATB-200, is more potent in and more efficient at targeting skeletal muscle tissues than conventional rhGAA such as Lumizyme®.
  • the rhGAA according to the invention has a superior ability to productively target muscle tissues in patients having Pompe Disease and reduce non-productive clearance of rhGAA as illustrated by Fig. 2.
  • the superior rhGAA according to the invention may be further completed or combined with chaperones or conjugated to other groups that target the CIMPR in muscle tissue, such as portions of IGF2 that bind to this receptor.
  • the Examples below show that the rhGAA of the invention, exemplified by ATB-200 rhGAA, exceeds the existing standard of care for enzyme replacement therapy by providing significantly better glycogen clearance in skeletal muscle as compared to existing regimen using the conventional rhGAA product Lumizyme®.
  • This application file contains at least one drawing executed in color.
  • Fig. 1 A shows a non-phosphorylated high mannose glycan, a mono-M6P glycan, and a bis-M6P glycan.
  • Fig. 1 B shows the chemical structure of the M6P group.
  • Fig. 2 Fig. 2 A describes productive targeting of rhGAA via glycans bearing M6P to target tissues (e.g., muscle tissues of subject with Pompe Disease).
  • Fig. 2B describes non-productive drug clearance to non-target tissues (e.g., liver and spleen) or by binding of non-M6P glycans to non-target tissues.
  • Fig. 3 Fig. 3 A graphically depicts a CIMPR receptor (also known as an IGF2 receptor) and domains of this receptor.
  • Fig. 3B is a table showing binding affinity (nMolar) of glycans bearing bis- and mono-M6P for CIMPR, the binding affinity of high mannose-type glycans to mannose receptors, and the binding affinity of de- sialyated complex glycan for asialyoglycoprotein receptors.
  • RhGAA that has glycans bearing M6P and bis-M6P can productively bind to CIMPR on muscle target cells.
  • RhGAA that has high mannose glycans and de-sialylated glycans can non-productively bind to non-target cells bearing the corresponding receptors.
  • Figs. 4A and 4B respectively, show the results of CIMPR affinity chromatography of Lumizyme® and Myozyme®.
  • the dashed lines refer to the M6P elution gradient. Elution with M6P displaces GAA molecules bound via an M6P- containing glycan to CIMPR.
  • Fig. 4A 78% of the GAA activity in Lumizyme® eluted prior to addition of M6P.
  • Fig. 4B shows that 73% of the GAA Myozyme® activity eluted prior to addition of M6P. Only 22% or 27% of the rhGAA in Lumizyme® or Myozyme, respectively, was eluted with M6P.
  • These figures show that most of the rhGAA in these two conventional rhGAA products lack glycans having M6P needed to target CIMPR in target muscle tissues.
  • a DNA construct for transforming CHO cells with DNA encoding rhGAA was transformed with a DNA construct encoding rhGAA (SEQ ID NO: 4).
  • Figs. 6A and 6B show the results of CIMPR affinity chromatography of Myozyme and ATB-200 rhGAA. As apparent from Fig. 6B, about 70% of the rhGAA in ATB-200 rhGAA contained M6P.
  • Fig. 8 Polywax elution profiles of Lumizyme® and ATB-200.rhGAAs.
  • Fig. 9 Summary of N-glycan structures of Lumizyme® compared to three different preparations of ATB200 rhGAA, identified as BP-rhGAA, ATB200-1 and ATB200-2.
  • Fig. 10 Fig. 10A compares the CIMPR binding affinity of ATB-200 rhGAA (left trace) with that of Lumizyme® (right trace).
  • Fig. 1 OB describes the Bis-M6P content of Lumizyme® and ATB-200 rhGAA.
  • Fig. 11 Fig. 1 1 A compares ATB-200 rhGAA activity (left trace) with Lumizyme® rhGAA activity (right trace) inside normal fibroblasts at various GAA concentrations.
  • Fig. 1 IB compares ATB-200 rhGAA activity (left trace) with Lumizyme® rhGAA activity (right trace) inside fibroblasts from a subject having Pompe Disease at various GAA concentrations.
  • Fig. 11C compares (K upt ake) of fibroblasts from normal subjects and subjects with Pompe Disease.
  • Fig. 12 Fig. 12A shows the amount of glycogen relative to protein in heart muscle after contact with vehicle (negative control), with 20 mg/ml Alglucosidase alfa, or with 5, 10 or 20 mg/kg ATB-200 rhGAA.
  • Fig. 12B shows the amount of glycogen relative to protein in quadriceps muscle after contact with vehicle (negative control), with 20 mg/ml Lumizyme®, or with 5, 10 or 20 mg/kg ATB-200 rhGAA.
  • Fig. 12C shows the amount of glycogen relative to protein in triceps muscle after contact with vehicle (negative control), with 20 mg/ml Lumizyme®, or with 5, 10 or 20 mg/kg ATB-200 rhGAA.
  • ATB-200 rhGAA produced significant glycogen reductions in quadriceps and triceps muscle compared to the negative control and compared to Lumizyme®.
  • Fig. 13 ATB-200 rhGAA stability is improved in the presence of chaperone AT2221.
  • the first, left trace in Fig. 13A shows percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 7.4 (blood pH).
  • the last, right trace shows percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 5.2
  • Fig. 14 This table shows that the combination of ATB-200 rhGAA and chaperone AT2221 provided significantly better glycogen clearance in GAA knock-out mice than treatments with Lumizyme® and AT2221 or controls of either Lumizyme® or ATB200 rhGAAs without the AT2221 chaperone.
  • Miglustat (AT2221) over those treated with ERT alone.
  • PAS glycogen staining (Fig. 16A) and EM (Fig. 16 B) of muscle tissue from GAA O mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221).
  • Fig. 16C Evaluation of lysosomal proliferation by LAMP-1 marker.
  • Fig. 16D Identification of Type I and Type II muscle fibers.
  • Miglustat (AT2221) over those treated with ERT alone.
  • PAS glycogen staining (Fig. 17A) of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and miglustat (AT-2221).
  • Fig. 17B Evaluation of lysosomal proliferation by LAMP-1 marker.
  • GAA refers to human acid a-glucosidase (GAA) an enzyme that catalyzes the hydrolysis of a- 1 ,4- and a-l,6-glycosidic linkages of lysosomal glycogen as well as to insertional, relational or substitution variants of the GAA amino acid sequence and fragments of a longer GAA sequence that exert enzymatic activity.
  • GAA human acid a-glucosidase
  • rhGAA is used to distinguish endogenous GAA from synthetic or recombinant-produced GAA, such as that produced by transformation of CHO cells with DNA encoding GAA.
  • An exemplary DNA sequence encoding GAA is NP 000143.2 (SEQ ID NO: 4) which is incorporated by reference.
  • GAA and rhGAA may be present in a composition containing a mixture of GAA molecules having different glycosylation patterns, such as a mixture of rhGAA molecules bearing mono-M6P or bis-M6P groups on their glycans and GAA molecules that do not bear M6P or bis-M6P.
  • GAA and rhGAA may also be completed with other compounds, such as chaperones, or may be bound to other moieties in a GAA or rhGAA conjugate, such as bound to an IGF2 moiety that targets the conjugate to CIMPR and subsequently delivers it to the lysosomes.
  • a “subject” or “patient” is preferably a human, though other mammals and non- human animals having disorders involving accumulation of glycogen may also be treated.
  • a subject may be a fetus, a neonate, child, juvenile or an adult with Pompe disease or other glycogen storage or accumulation disorder.
  • One example of an individual being treated is an individual (fetus, neonate, child, juvenile, adolescent, or adult human) having GSD-II (e.g., infantile GSD-II, juvenile GSD-II, or adult-onset GSD-II).
  • the individual can have residual GAA activity, or no measurable activity.
  • the individual having GSD-II can have GAA activity that is less than about 1% of normal GAA activity (infantile GSD-II), GAA activity that is about 1-10% of normal GAA activity (juvenile GSD-II), or GAA activity that is about 10-40% of normal GAA activity (adult GSD-II).
  • treat and “treatment,” as used herein, refer to amelioration of one or more symptoms associated with the disease, prevention or delay of the onset of one or more symptoms of the disease, and/or lessening of the severity or frequency of one or more symptoms of the disease.
  • treatment can refer to improvement of cardiac status (e.g.
  • treatment includes improvement of cardiac status, particularly in reduction or prevention of GSD-II-associated cardiomyopathy.
  • improve indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
  • a control individual is an individual afflicted with the same form of GSD-II (either infantile, juvenile or adult-onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).
  • purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i. e. , contaminants, including native materials from which the material is obtained.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 95% pure; more preferably, at least 97% pure, and more preferably still at least 99% pure.
  • purified can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, enzymatic assay and other methods known in the art.
  • purified means that the level of contaminants is below a level acceptable to regulatory authorities for safe administration to a human or non-human animal.
  • Recombinant proteins may be isolated or purified from CHO cells using methods known in the art including by chromatographic size separation, affinity chromatography or anionic exchange chromatography.
  • genetically modified refers to cells, such as CHO cells, that express a particular gene product, such as rhGAA or ATB-200 rhGAA, following introduction of a nucleic acid comprising a coding sequence which encodes the gene product, along with regulatory elements that control expression of the coding sequence.
  • Introduction of the nucleic acid may be accomplished by any method known in the art including gene targeting and homologous recombination.
  • the term also includes cells that have been engineered to express or overexpress an endogenous gene or gene product not normally expressed by such cell, e.g. , by gene activation technology.
  • Pompe Disease refers to an autosomal recessive LSD characterized by deficient acid alpha glucosidase (GAA) activity which impairs lysosomal glycogen metabolism.
  • GAA acid alpha glucosidase
  • the enzyme deficiency leads to lysosomal glycogen accumulation and results in progressive skeletal muscle weakness, reduced cardiac function, respiratory insufficiency, and/or CNS impairment at late stages of disease.
  • Genetic mutations in the GAA gene result in either lower expression or produce mutant forms of the enzyme with altered stability, and/or biological activity ultimately leading to disease, (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid a-Glucosidase (Acid Maltase) Deficiency, The Metabolic and
  • Pompe Disease infantile, juvenile and adult
  • the three recognized clinical forms of Pompe Disease are correlated with the level of residual a-glucosidase activity (Reuser A J et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61- S69).
  • Infantile Pompe disease type I or A
  • Juvenile Pompe disease type II or B
  • Juvenile Pompe disease is intermediate in severity and is characterized by a predominance of muscular symptoms without
  • the term "Pompe Disease” refers to all types of Pompe Disease.
  • the formulations and dosing regimens disclosed in this application may be used to treat, for example, Type I, Type II or Type III Pompe Disease.
  • An exemplary rhGAA composition according to the invention is ATB-200 (sometimes designated ATB-200, ATB-200 or CBP-rhGAA) which is described in the Examples.
  • the rhGAA of the invention (ATB-200) has been shown to bind the CIMPR with high affinity (KD ⁇ 2-4 nM) and to be efficiently internalized by Pompe fibroblasts and skeletal muscle myoblasts (K up take ⁇ 7-14 nM).
  • ATB-200 was characterized in vivo and shown to have a shorter apparent plasma half-life (ty 2 ⁇ 45 min) than the current rhGAA ERT (ti/ 2 ⁇ 60 min).
  • the amino acid sequence of the rhGAA can be at least 70%, 75%, 80%, 85%, 95% or
  • the GAA or rhGAA of the invention will comprise a wild-type GAA amino acid sequence such as that of SEQ ID NO: 1 or 3.
  • the rhGAA comprises a subset of the amino acid residues present in a wild-type GAA, wherein the subset includes the amino acid residues of the wild-type GAA that form the active site for substrate binding and/or substrate reduction.
  • the rhGAA is glucosidase alfa, which is the human enzyme acid a- glucosidase (GAA), encoded by the most predominant of nine observed haplotypes of this gene.
  • the rhGAA of the invention including ATB-200 rhGAA, may comprise an amino acid sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of human alpha glucosidase, such as that given by accession number AHE24104.1 (GI:568760974)(SEQ ID NO: 1) and which is incorporated by reference to U.S. Patent No. 8,592,362 or to the amino acid sequence of NP 000143.2 (SEQ ID NO: 4).
  • a nucleotide and amino acid sequence for GAA is also given by SEQ ID NOS: 2 and 3, respectively. Variants of this amino acid sequence also include those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more amino acid deletions, insertions or substitutions to the GAA amino acid sequence below. Polynucleotide sequences encoding GAA and such variant human GAAs are also provided.
  • rhGAAs contemplated and may be used to recombinantly express rhGAAs according to the invention.
  • Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.
  • FASTA Altschul et al.
  • BLAST Garnier et al.
  • polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62.
  • BLASTP When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score.
  • BLASTP "Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other.
  • Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure.
  • the polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.
  • no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or 5% of the total rhGAA in the composition according to the invention lacks an N-glycan bearing M6P or bis-M6P or lacks a capacity to bind to the cationic independent manose-6-phosphate receptor (CIMPR).
  • CIMPR cationic independent manose-6-phosphate receptor
  • 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99%, ⁇ 100% or more of the rhGAA in the composition comprises at least one N-glycan bearing M6P and/or bis-M6P or has the capacity to bind to CIMPR.
  • the rhGAA molecules in the rhGAA composition of the invention may have 1, 2, 3 or 4 M6P groups on their glycans.
  • M6P mono-phosphorylated
  • a single N-glycan may bear two M6P groups (bis- phosphorylated)
  • two different N-glycans on the same rhGAA molecule may bear single M6P groups.
  • rhGAA molecules in the rhGAA composition may also have N-glycans bearing no M6P groups.
  • the N-glycans contain greater than 3 mol/mol of M6P and greater than 4 mol/mol sialic acid.
  • the total glycans on the rhGAA may be in the form of a mono-M6P glycan, for example, about 6.25% of the total glycans may carry a single M6P group and on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans on the rhGAA are in the form of a bis-M6P glycan and on average less than 25% of total rhGAA of the invention contains no phosphorylated glycan binding to CIMPR.
  • the rhGAA composition according to the invention may have an average content of N-glycans carrying M6P ranging from 0.5 to 7.0 mol/mol rhGAA or any intermediate value of subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mol/mol rhGAA.
  • the rhGAA of the invention can be fractionated to provide rhGAA compositions with different average numbers of M6P-bearing or bis-M6P- bearing glycans on the rhGAA thus permitting further customization of rhGAA targeting to the lysosomes in target tissues by selecting a particular fraction or by selectively combining different fractions.
  • Up to 60% of the N-glycans on the rhGAA may be fully sialyated, for example, up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans may be fully sialyated. In some embodiments from 4 to 20% of the total N-glycans in the rhGAA composition are fully sialylated.
  • no more than 5%, 10%, 20% or 30% of N-glycans on the rhGAA carry sialic acid and a terminal Gal.
  • This ranges includes all intermediate values and subranges, for example, 7 to 30% of the total N-glycans on the rhGAA in the composition can carry sialic acid and terminal Gal.
  • no more than 5, 10, 15, 16, 17, 18, 19 or 20% of the N- glycans on the rhGAA have a terminal Gal only and do not contain sialic acid.
  • This range includes all intermediate values and subranges, for example, from 8 to 19% of the total N- glycans on the rhGAA in the composition may have terminal Gal only and do not contain sialic acid.
  • the total N-glycans on the rhGAA in the composition are complex type N-glycans; or no more than 1, 2, 3, 4, 5, 6, 7% of total N-glycans on the rhGAA in the composition are hybrid-type N-glycans; no more than 5, 10, or 15% of the high mannose-type N-glycans on the rhGAA in the composition are non-phosphorylated; at least 5% or 10% of the high mannose-type N-glycans on the rhGAA in the composition are mono-M6P phosphorylated; and/or at least 1 or 2% of the high mannose-type N-glycans on the rhGAA in the composition are bis-M6P phosphorylated.
  • These values include all intermediate values and subranges.
  • An rhGAA composition according to the invention may meet one or more of the content ranges described above.
  • the rhGAA composition of the invention will bear, on average,
  • sialic acid residues may prevent non-productive clearance by asialoglycoprotein receptors.
  • the rhGAA composition of the invention is preferably produced by CHO cells, such as CHO cell line GA-ATB-200, or by a subculture or derivative of such a CHO cell culture.
  • DNA constructs which express allelic variants of GAA or other variant GAA amino acid sequences such as those that are at least 90%, 95% or 99% identical to SEQ ID NO: 1, may be constructed and expressed in CHO cells.
  • Those of skill in the art can select alternative vectors suitable for transforming CHO cells for production of such DNA constructs.
  • rhGAA having superior ability to target the CIMPR and cellular lysosomes as well as glycosylation patterns that reduce its non-productive clearance in vivo can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express rhGAA with significantly higher levels of total M6P and bis-M6P than conventional rhGAA products.
  • the recombinant human GAA produced by these cells has significantly more muscle cell-targeting M6P and bis-M6P groups than conventional GAA, such as Lumizyme® and has been shown to efficiently bind to CIMPR and be efficiently taken up by skeletal muscle and cardiac muscle. It has also been shown to have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces non-productive clearance in vivo.
  • the rhGAA according to the invention may be formulated as a pharmaceutical composition or used in the manufacture of a medicament for treatment of Pompe Disease or other conditions associated with a deficient of GAA.
  • the compositions can be formulated with a physiologically acceptable carrier or excipient.
  • the carrier and composition can be sterile and otherwise suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g. , NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g.
  • surfactants such as polysorbates like polysorbate 80, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not
  • a water-soluble carrier suitable for intravenous administration is used.
  • the composition or medicament can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the rhGAA is administered by IV infusion.
  • composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings.
  • a composition for intravenous administration is a solution in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage faun, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the rhGAA can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • rhGAA (or a composition or medicament containing GAA) is administered by an appropriate route.
  • the GAA is administered intravenously.
  • GAA is administered by direct administration to a target tissue, such as to heart or skeletal muscle (e.g., intramuscular), or nervous system (e.g. , direct injection into the brain; intraventricular ly; intrathecally). More than one route can be used concurrently, if desired.
  • the rhGAA (or a composition or medicament containing GAA) is administered in a therapeutically effective amount (e.g. , a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease, as described above).
  • a therapeutically effective amount e.g. , a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease, as described above.
  • the amount which will be therapeutically effective in the treatment of the disease will depend on the nature and extent of the disease's effects, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the therapeutically effective amount is equal of less than 20 mg enzyme/kg body weight of the individual, preferably in the range of about 1-10 mg enzyme/kg body weight, and even more preferably about 10 mg enzyme/kg body weight or about 5 mg enzyme/kg body weight.
  • the effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if anti-GAA antibodies become present or increase, or if disease symptoms worsen, the amount can be increased.
  • the therapeutically effective amount of GAA (or composition or medicament containing GAA) is administered at regular intervals, depending on the nature and extent of the disease's effects, and on an ongoing basis.
  • Administration at a "regular interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose).
  • the interval can be determined by standard clinical techniques.
  • GAA is administered monthly, bimonthly; weekly; twice weekly; or daily.
  • the administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual. For example, in times of physical illness or stress, if anti-GAA antibodies become present or increase, or if disease symptoms worsen, the interval between doses can be decreased. In some
  • a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme/kg body weight is administered twice a week, weekly or every other week with or without a chaperone.
  • the GAA or rhGAA of the invention may be prepared for later use, such as in a unit dose vial or syringe, or in a bottle or bag for intravenous administration.
  • Kits containing the GAA or rhGAA, as well as optional excipients or other active ingredients, such as chaperones or other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution or dosing for treating a subject in need of treatment, such as a patient having Pompe disease.
  • GAA (or a composition or medicament containing GAA) can be administered alone, or in conjunction with other agents, such as a chaperone.
  • rhGAA with different degrees of glycosylation with mono-M6P or bis-M6P may be administered or combinations of rhGAAs with different degrees of M6P or bisM6P glycosylate administered.
  • the rhGAA composition of the invention will be complexed or admixed with a chaperone, such as AT-2220 or AT-2221.
  • Chaperones sometimes referred to as “pharmacological chaperones,” are compounds that when complexed or coadministered with rhGAA modify its pharmacokinetics and other pharmacological properties.
  • chaperones exemplified herein include AT2221 (miglustat, N-butyl- deoxynojirimycin) and AT2220 (duvoglustat HCl, 1-deoxynojirimycin). Such complexing or admixing may occur outside the body or inside the body, for example, where separate dosages of the rhGAA and chaperone are administered.
  • targeting of active rhGAA, its fractions, or derivatives of the invention to CIMPR and subsequently to cellular lysosomes may be improved by combining it duvoglustat-HCl (AT2220, deoxynojirimycine, AT2220) or miglustat (AT2221, N-butyl-deoxynojirimycin).
  • the Examples below show significant glycogen substrate reductions in key skeletal muscles of GAA-knock-out mice receiving the well-targeted rhGAA of the invention in combination with a chaperone.
  • Another aspect of the invention pertains to CHO cells or their derivatives or other equivalents that produce the rhGAA according to the invention.
  • a CHO cell line is GA-ATB-200 or a subculture thereof that produces a rhGAA composition as described herein.
  • Such CHO cell lines may contain multiple copies of a gene, such as 5, 10, 15, or 20 or more copies, of a polynucleotide encoding GAA.
  • the high M6P and bis-M6P rhGAA of the invention can be produced by transforming CHO cells (Chinese hamster ovary cells) with a DNA construct that encodes GAA. While CHO cells have been previously used to make rhGAA, it was not appreciated that transformed CHO cells could be cultured and selected in a way that would produce rhGAA having a high content of M6P and bis-M6P glycans which target the
  • a related aspect of the invention is directed to method for making these CHO cell lines.
  • This method involves transforming a CHO cell with DNA encoding GAA or a GAA variant, selecting a CHO cell that stably integrates the DNA encoding GAA into its chromosome(s) and that stably expresses GAA, and selecting a CHO cell that expresses GAA having a high content of glycans bearing M6P or bis-M6P, and, optionally, selecting a CHO cell having N-glycans with high sialic acid content and/or having N-glycans with a low non-phosphorylated high- mannose content.
  • CHO cell lines may be used to produce rhGAA and rhGAA compositions according to the invention by culturing the CHO cell line and recovering said composition from the culture of CHO cells.
  • the rhGAA composition of the invention or its fractions or derivatives is
  • a subject in need of treatment includes those having Glycogen Storage Disease Type ⁇ (Pompe Disease) as well as other conditions, disorders or diseases which would benefit from the administration of the rhGAA.
  • the rhGAA of the invention (ATB-200) is taken up by skeletal muscle cells, binds to CIMPR and effectively removes glycogen from skeletal muscle cells when administered at a significantly lower dosage than conventional rhGAA products.
  • a reduction of up to 75% of glycogen in skeletal muscle myoblast was attained in GAA-knockout mice using a biweekly regimen of intravenous administration of ATB-200.
  • These reductions exceeded those provided by the same amount of Lumizyme® showing that the rhGAA of the invention, which has an enhanced content of N-glycans bearing M6P and bis-M6P, provided superior reductions in glycogen substrate.
  • Due to the improved targeting, pharmacodynamics and pharmacokinetics of the rhGAA composition of the invention may be administered in a lower dosage than conventional rhGAA products such as Lumizyme® or Myozyme®.
  • skeletal or striated muscles subject to treatment include at least one muscle selected from the group consisting of abductor digiti minimi (foot), abductor digiti minimi (hand), abductor halluces, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor halluces, adductor longus, adductor magnus, adductor pollicis, anconeus, articularis cubiti, articularis genu, aryepiglotticus,
  • aryjordanicus auricularis, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinators, bulbospongiosus, constrictor of pharynx-inferior, constrictor of pharynx- middle, constrictor of pharynx-superior, coracobrachialis, corrugator supercilii, cremaster, cricothyroid, dartos, deep transverse perinei, deltoid, depressor anguli oris, depressor labii inferioris, diaphragm, digastric, digastric (anterior view), erector spinae- spinalis, erector spinae-iliocostalis, erector spinae-longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ul
  • s -semispinalis transversus abdominis, transversus thoracis, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor.
  • the GAA composition of the invention may also be administered to, or used to treat, type 1 (slow twitch) muscle fiber or type 2 (fast twitch) muscle fiber or subjects accumulating glycogen in such muscle fibers.
  • type 1 slow twitch
  • type 2 fast twitch
  • Type I slow twitch, or "red” muscle
  • Slow twitch fibers contract for long periods of time but with little force.
  • Type II, fast twitch muscle has three major subtypes (Ila, IIx, and lib) that vary in both contractile speed and force generated.
  • Fast twitch fibers contract quickly and powerfully but fatigue very rapidly, sustaining only short, anaerobic bursts of activity before muscle contraction becomes painful. They contribute most to muscle strength and have greater potential for increase in mass.
  • Type lib is anaerobic, glycolytic, "white” muscle that is least dense in mitochondria and myoglobin. In small animals (e.g. , rodents) this is the major fast muscle type, explaining the pale color of their flesh.
  • the rhGAA composition of the invention may be administered systemically, for example, by intravenous (IV) infusion, or administered directly into a desired site, such as into cardiac or skeletal muscle, such as quadriceps, triceps, or
  • diaphragm It may be administered to myocytes, particular muscle tissues, muscles, or muscle groups.
  • myocytes particular muscle tissues, muscles, or muscle groups.
  • such a treatment may administer intramuscularly the rhGAA composition directly into a subject's quadriceps or triceps or diaphragm.
  • the rhGAA composition of the invention can be complexed or admixed with a chaperone, such as AT-2220 (Duvoglustat HQ, 1-Deoxynojirimycin) or AT-2221 ( iglustat, N-butyl-deoxynojirimycin) or their salts to improve the pharmacokinetics of the rhGAA administration.
  • a chaperone such as AT-2220 (Duvoglustat HQ, 1-Deoxynojirimycin) or AT-2221 ( iglustat, N-butyl-deoxynojirimycin) or their salts to improve the pharmacokinetics of the rhGAA administration.
  • the rhGAA and the chaperone may be administered together or separately.
  • the GAA in the composition may be preloaded with the chaperone.
  • the GAA and the chaperone may be administered separately either at the same time or at different times.
  • Representative dosages of AT2221 range from 0.25 to 400 mg/kg, preferably from 0.5-200 mg/kg, and most preferably from 2 to 50 mg/kg.
  • Specific dosages of AT2221 include 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mg/kg.
  • These dosages may be combined with rhGAA, such as ATB-200 rhGAA, at a molar ratio of AT2221 to rhGAA ranging from 15: 1 to 150: 1.
  • ratios include 15: 1 , 20: 1 , 25: 1 , 50: 1 , 60: 1 , 65: 1 , 70: 1 , 75: 1 , 80: 1 , 85: 1 , 90: 1 , 100: 1 , 125: 1 , and 150: 1.
  • rhGAA and AT2221 may be coadministered in these amounts or molar ratios either concurrently, sequentially or separately.
  • the ranges above include all intermediate subranges and values, such as all integer values between the range endpoints.
  • Representative dosages of AT2220 range from 0.1 to 120 mg/kg, preferably 0.25 to 60, and most preferably from 0.6 to 15 mg/kg.
  • Specific dosages of AT2220 include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 and 30 mg/kg. These dosages may be combined with rhGAA, such as ATB-200 rhGAA, at a molar ratio of AT2220 to rhGAA ranging from 15 : 1 to 150: 1.
  • Specific ratios include 15 : 1, 20: 125: 1 , 50: 1 , 60: 1 , 65: 1 , 70: 1, 75: 1, 80: 1, 85: 1 , 90: 1 , 100: 1, 125: 1 , and 150: 1.
  • rhGAA and AT2220 may be coadministered in these amounts or molar ratios either concurrently, sequentially or separately.
  • the ranges above include all intermediate subranges and values, such as all integer values between the range endpoints.
  • the rhGAA composition of the invention, its fractions or derivatives may also be used for metabolizing, degrading, removing or otherwise decreasing glycogen in tissue, muscle, muscle fiber, muscle cells, lysosomes, organelles, cellular compartments, or cytoplasm.
  • the rhGAA of the invention may be used for modulating lysosomal proliferation, autophagy, or exocytosis in a cell by administering it, its fractions, or derivatives to cells, tissues, or subjects in need of such modulation, optionally in combination with a chaperone or optionally as a conjugate with another targeting moiety.
  • Autophagy is a catabolic mechanism that allows a cell to degrade glycogen or other unnecessary or dysfunctional cellular components through the actions of it lysosomes. This method can also involve systemically or locally administering the GAA composition to a subject in need of treatment.
  • the rhGAA according to the invention which is enriched for mono-M6P and bis- M6P, compared to Lumizyme® and Myozyme, and which has favorable pharmacokinetic properties conferred by its glycosylation pattern may also be used for treatment of other conditions requiring the breakdown of complex carbohydrates, such as other disorders in which glycogen or other carbohydrates degraded by rhGAA accumulate in the lysosomes or other parts of the cell, such as in the cytoplasm accessible to rhGAA, such as Glycogen storage disease III. It may also be used non-therapeutic purposes, such as for the production of foods, beverages, chemicals and pharmaceutical products which require breaking down complex carbohydrates such as starch and glycogen into their monomers.
  • Section I ATB-200 rhGAA and its properties
  • Fig. 5 describes the problems associated with conventional ERTs (Myozyme® and Lumizyme®): 73% of the rhGAA in Myozyme® (Fig. 5B) and 78% of the rhGAA in Lumizyme® (Fig. 5A) did not bind to the CIMPR, see the left-most peaks in each figure. Only 27% of the rhGAA in Myozyme® and 22% of the rhGAA in Lumizyme® contained M6P that can productive target it to the CIMPR on muscle cells, see Fig. 2 which describes productive drug targeting and non-productive drug clearance.
  • An effective dose of Myozyme® and Lumizyme® corresponds to the amount of rhGAA containing M6P which targets the CIMPR on muscle cells.
  • most of the rhGAA in these two conventional products does not target the CIMPR receptor on target muscle cells.
  • the administration of a conventional rhGAA where most of the rhGAA is not targeted to muscle cells increases the risk of allergic reaction or induction of immunity to the non-targeted rhGAA.
  • CHO cells were transfected with DNA that expresses rh-GAA followed by selection of transformants producing rhGAA.
  • a DNA construct for transforming CHO cells with DNA encoding rh-GAA is shown in Fig. 5.
  • CHO cells were transfected with DNA that expresses rh-GAA followed by selection of transformants producing rhGAA.
  • DG44 CHO (DHFR-) cells containing a stably integrated GAA gene were selected with hypoxanthine/thymidine deficient (-HT) medium). Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools that expressed high amounts of GAA were identified by GAA enzyme activity assays and were used to establish individual clones producing rhGAA. Individual clones were generated on semisolid media plates, picked by ClonePix system, and were transferred to 24- deep well plates. The individual clones were assayed for GAA enzyme activity to identify clones expressing a high level of GAA.
  • MTX methotrexate treatment
  • Conditioned media for determining GAA activity used a 4-MU-a-Glucosidase substrate. Clones producing higher levels of GAA as measured by GAA enzyme assays were further evaluated for viability, ability to grow, GAA productivity, N-glycan structure and stable protein expression.
  • CHO cell lines including CHO cell line GA-ATB-200, expressing rhGAA with enhanced mono-M6P or bis-M6P N- glycans were isolated using this procedure.
  • ATB- 200 rhGAA was obtained from CHO cells and purified.
  • Lumizyme® was obtained from a commercial source. Lumizyme® exhibited a high peak on the left of its elution profile.
  • ATB- 200 rhGAA exhibited four prominent peaks eluting to the right of Lumizyme® (Fig. 8). This confirms that ATB-200 rhGAA was phosphorylated to a greater extent than Lumizyme® since this evaluation is by terminal charge rather than CIMPR affinity.
  • ATB-200 rhGAA and Lumizyme® glycans were evaluated by MALDI-TOF to determine the individual glycan structures found on each ERT (Fig. 9).
  • ATB-200 samples were found to contain slightly lower amounts of non-phosphorylated high-mannose type N- glycans than Lumizyme®.
  • Lumizyme targets ATB-200 rhGAA to muscle cells more effectively.
  • the high percentage of mono-phosphorylated and bis-phosphorylated structures determined by MALDI agree with the CIMPR profiles which illustrated significantly greater binding of ATB-200 to the CIMPR receptor.
  • N-glycan analysis via MALDI-TOF mass spectrometry confirmed that on average each ATB200 molecule contains at least one natural bis-M6P N-glycan structure. This higher bis-M6P N-glycan content on ATB-200 rhGAA directly correlated with high-affinity binding to CIMPR in M6P receptor plate binding assays (KD about 2-4 nM) Figure 10A. Characterization of CIMPR Affinity of ATB-200
  • Lumizyme® and ATB200 rhGAA receptor binding was determined using a CIMPR plate binding assay. Briefly, CIMPR- coated plates were used to capture GAA. Varying concentrations of rhGAA were applied to the immobilized receptor and unbound rhGAA was washed off. The amount of remaining rhGAA was determined by GAA activity. As shown by Fig. 10A, ATB-200 rhGAA bound to CIMPR significantly better than Lumizyme.
  • Fig. 10B shows the relative content of bis-M6P glycans in Lumizyme, a conventional rhGAA, and ATB-200 according to the invention.
  • Lumizyme® there is on average only 10% of molecules have a bis-phosphorylated glycan. Contrast this with ATB-200 where on average every rhGAA molecule has at least one bis-phosphorylated glycan.
  • ATB-200 rhGAA was more efficiently internalized by fibroblast than Lumizyme.
  • the relative cellular uptake of ATB-200 and Lumizyme® rhGAA were compared using normal and Pompe fibroblast cell lines. Comparisons involved 5-100 nM of ATB-200 rhGAA according to the invention with 10-500 nM conventional rhGAA Lumizyme®. After 16-hr incubation, external rhGAA was inactivated with TRIS base and cells were washed 3- times with PBS prior to harvest. Internalized GAA measured by 4MU-a-Glucoside hydrolysis and was graphed relative to total cellular protein and the results appear in Fig. 11.
  • ATB-200 rhGAA was also shown to be efficiently internalized into cells (Figure 1 1 A and 1 IB), respectively, show that ATB-200 rhGAA is internalized into both normal and Pompe fibroblast cells and that it is internalized to a greater degree than conventional Lumizyme® rhGAA.
  • ATB-200 rhGAA saturates cellular receptors at about 20 nM, while about 250 nM of Lumizyme® is needed.
  • the uptake efficiency constant (K uptake ) extrapolated from these results is 2-3 nm for ATB-200 and 56 nM for Lumizyme® as shown by Fig. l lC.
  • ERT enzyme replacement therapy
  • rhGAA recombinant human GAA
  • CIMPRs cell surface cation-independent M6P receptors
  • the inventors developed a production cell line and manufacturing process that yield rhGAA (designated as ATB-200 rhGAA) with superior glycosylation and higher M6P content than conventional rhGAA, particularly the high-affinity bis-M6P N-glycan structure, for improved drug targeting.
  • ATB-200 rhGAA binds the CI-MPR with high affinity (KD ⁇ 2-4 nM) and was efficiently internalized by Pompe fibroblasts and skeletal muscle myoblasts (K upt ake—7-14 nM).
  • ATB-200 rhGAA clears glycogen significantly better than Lumizyme® in skeletal muscle.
  • the effects of administering Lumizyme® and ATB-200 rhGAA for glycogen clearance in GAA KO mice were evaluated. Animals were given two IV bolus
  • ATB-200 rhGAA and Lumizyme® rhGAA were equally effective for clearing glycogen in heart (Fig 12A). As show in in Figs. 12B and 12C, ATB-200 rhGAA at 5 mg/kg was equivalent to Lumizyme® rhGAA at 20 mg/kg for reducing glycogen in skeletal muscles; ATB-200 dosed at 10 and 20 mg/kg was significantly better than Lumizyme® for clearing glycogen in skeletal muscles;
  • a chaperone binds to and stabilizes rhGAA ERT, increases uptake of active enzyme into tissues, improves tolerability and potentially mitigates immunogenicity. As shown above, the protein stability of ERT under unfavorable conditions was substantially improved using CHARTTM. CHART: chaperone-advanced replacement therapy, see
  • Figs. 13A and 13B the stability of ATB-200 was significantly improved by AT2221 (Miglustat, N-butyl-deoxynojirimycin). Folding of rhGAA protein was monitored at 37 °C by thermal denaturation in neutral (pH 7.4 - plasma environment) or acidic (pH 5.2 - lysosomal environment) buffers. AT2220 stabilized rhGAA protein in neutral pH buffer over 24 hours. Co-administration of Myozvme® with AT2221 (Miglustat) Compared to Coadministration of ATB-200 rhGAA with Miglustat
  • the combination of a pharmacological chaperone and ATB-200 rhGAA was found to enhance glycogen clearance in vivo.
  • GAA KO mice were given two IV bolus administrations of rhGAA at 20 mg/kg every other week.
  • the pharmacological chaperone AT2221 was orally administered 30 mins prior to rhGAA at dosages of 0, 1, 2 and 10 mg/kg.
  • Tissues were harvested two weeks after the last dose of ERT and analyzed for GAA activity, glycogen content cell specific glycogen and lysosome proliferation.
  • the animals receiving ATB200+ chaperone AT2221 exhibited enhanced glycogen clearance from quadriceps muscle.
  • ATB-200 rhGAA (20 mg/kg) reduced glycogen more than the same dose of Lumizyme® and when ATB-200 rhGAA was combined with 10 mg/kg of AT2220 near normal levels of glycogen in muscle were attained.
  • Figs. 16A and 16B unlike conventional rhGAA, which showed limited glycogen reduction (indicated by abundant punctate PAS signal), ATB-200 rhGAA alone showed a significant decrease in PAS signals. Co-administration with 10 mg/kg miglustat resulted in a substantial further reduction in substrate.
  • ATB-200 rhGAA which has higher levels of M6P and bis- M6P on its N-glycans efficiently targets CIMPR in skeletal muscle.
  • ATB-200 rhGAA also has well-processed complex-type N-glycans that minimize non-productive clearance in vivo, has pharmacokinetic properties favorable for its use in vivo and exhibits good targeting to key muscle tissues in vivo.
  • ATB-200 rhGAA is better than the conventional standard of care, Lumizyme, for reducing glycogen in muscle tissue and that a combination of ATB-200 rhGAA and chaperone AT2221 further improve removal of glycogen from target tissues and improves muscle pathology.
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